Collagen is defined as protein or glycoprotein having a helical structure (collagen helices) at least partially. This is a triple helix comprising three polypeptide chains and, in each polypeptide chain of a molecular weight of about 100,000, a glycine residue appears every three amino acid residues and, as for other amino acid residues, proline residues and hydroxyproline residues appear frequently. Collagen can be extracted mainly from tissues, particularly from skin, of invertebrate or vertebrate animals. 19 generic types of collagens, classified by molecule structure, have been reported and in some cases, one collagen type so classified includes several different molecule species.
Particularly, collagens of types I, II, III and IV are mainly used as starting materials for biomaterials. Type I is present inmost of connective tissues and is a collagen type which is most abundantly present in living organism. Type I is especially abundant in tendons, coria and bones and, collagens for industrial uses are extracted from those sites in many cases. Type II is collagen which forms cartilage. Type III is often present in the same site as the type I although its amount is small. Type IV is collagen which forms basement membrane. Types I, II and III are present in living organism as collagen fibers and mainly play a role of maintaining the strength of tissues or organs. Although type IV cannot form fiber, it is said to form a network assembly of four molecules and to participate in cell differentiation in basement membranes. The term “collagen(s)” used in the present specification as hereinafter means collagen of type I, II or III or a mixture of two or more types thereof.
Collagen fiber is a self-aggregate of the above-mentioned collagen and has a specific fiber structure in which collagen molecules are packed in series and also in parallel. Industrially, soluble collagen is manufactured from collagen fiber in tissues using acid, alkali or proteinase.
Soluble collagen comprises fine assemblies consisting of not more than several collagen molecules, whereby can form a uniform and transparent solution when dissolved in water or in an aqueous salt solution. It is known that collagen molecules, once solubilized, can recreate collagen fiber in vitro under certain conditions. Such a phenomenon is called fibril formation or fibrillation and its properties are described in detail in Biochemical Journal, 316, pages 1-11 (1996).
When collagen is heated, the triple helical structure of collagen is raveled out and each polypeptide chain gives a thermally denatured product in a random coil form. The temperature causing such a structural change is called a denaturing temperature and such a thermally denatured product is called gelatin. It is known that, as compared with collagen, gelatin has a high solubility in water and a high sensitivity to protease in living organisms. It is also known that, depending upon the condition concerning solvent, gelatin partially can recover the collagen helical structure and that although ability for forming collagen fiber in collagen has been lost in the denaturing process, by thus partially recovering the collagen helical structure in gelatin, the ability for formation of collagen fiber can also be recovered.
Denaturing temperature of collagen is lowest in a state of solution. It is also said that, although collagen is usually obtained from living materials, denaturing temperature of collagen obtained from living organisms is closely related to the temperature of living environment of the living organism. Denaturing temperature of collagen of mammals in an aqueous solution is about 38° C. while denaturing temperature of collagen of fishes is generally lower than that of mammals and particularly, in some cases of collagens of fishes in cold currents such as salmon, the denaturing temperature is less than 20° C.
Collagen, for its excellent properties such as property of promoting adhesion and growth of cells, low antigenic property, high affinity for living organisms and biodegradable property, is advantageously used in various uses such as materials for cell experiments and medical materials. When used for such purposes, collagen is formed into various forms such as cotton-like product, film, sponge and gel depending upon the use. As preferable examples, for hemostatic material, collagen is used in form of cotton-like product, and for artificial skin, in form of sponge. Further, for cell experiments material, collagen is used in form of gel. However, collagen materials as such are usually in an aqueous state, fragile and less stretchable and therefore, in some cases, there are some limitations on application of such material to uses in cell experiment materials and medical materials.
For example, in recent years, it has been pointed out that characteristics of cells in an ordinary static incubation system (in vitro) are different in many respects from those in the system which receives mechanical stimulation in vivo and there has been an increasing demand for a cell experiment apparatus which gives mechanical stimulation easily and simply. For such a cell experiment apparatus, a cell carrier having both cell adhesion and stretching property is necessary and, for example, silicone membrane where fibronectin as a cell adhesion protein is coated is used (Am. J. Physiol. 274 (5 Pt 2), H 1532-1538 (1998)). However, many cells use collagen as a main footing in vivo and, in order to endow an in vivo-like environment to such cells, it is preferred to prepare a cell carrier using collagen. However, a stretching property is poor in conventional collagen materials and application of such a material to cell experiment apparatuses where mechanical stimulation is given is difficult.
In artificial skin for example, collagen sponge is favorably used for the purpose of endowing an environment suitable for healing the wound site to thereby promote tissue repair. However, conventional collagen sponge is poor in its stretching property and, when applied to wound sites on or around joints, the sponge is broken in some cases. Moreover, such a collagen sponge lacks strength for suture when applied to the wound site, therefore use of such a sponge involves troubles of using synthetic polymer in combination (JP-A-2001-104346) and the like. Furthermore, such a synthetic polymer is necessarily removed after the wound is healed and, at that time, the cured site receives some damages again.
As for artificial blood vessel, for example, in consideration for biocompatibility and antithrombotic property, an artificial blood vessel model of a hybrid type with an artificial tubular structure containing smooth muscle and having a flat lumen surface whereon a layer of endothelial cells may be formed.
As a specific model thereof, a model formed by molding gel-like collagen with smooth muscle mixed therein into a tubular structure is proposed (Science, 231, pages 397-400 (1986); ASAIO Journal, pages 383-388 (1994)). According to this model, artificial blood vessel having a flat lumen surface can be formed within a short period of time. However, such a product of tubular structure is fragile, and its strength is so low that the product immediately after manufactured may be broken if picked up with tweezers, and therefore there is a problem that such an artificial blood vessel cannot endure biomechanical environment present in living organisms.
In view of the above, another model is also proposed in which a culture liquid containing smooth muscle cells is directly sown on a biodegradable or non-biodegradable tubular structure having a relatively high mechanical strength and, after cultured until the lumen surface becomes flat, endothelial cells are sown (JP-A-2001-78750 (European Patent Laid-Open No. 1,214,952)). Such a model has a good mechanical strength and can be used as artificial blood vessel even for an artery. However, it is known that biodegradable or non-biodegradable tubular structure has a strong hydrophobicity and that its properties for adhesion and growth of cells are significantly bad. Therefore, there is a problem that it takes a long period of times of several months to culture the smooth muscle cells in the tubular structure until a flat lumen surface is formed. Such a problem makes the proposed model impractical in light of the situation where patients need artificial blood vessels. In addition, in the model, there remains another problem that, due to its low stretching property, abrasion takes place between the artificial vessel and inherent blood vessel after transplantation, a break is resulted at the bonding area and blood may leak out therefrom.
The above-mentioned problems are expected to be solved by imparting both stretching property and high mechanical strength to a collagen material. However, no collagen material having such properties and production method therefor has been disclosed.
Moreover, conventionally, most collagen serving as starting material for collagen materials, is collected from tissues of livestock, such as oxhide. However, BSE (bovine spongiform encephalopathy) have emerged in recent years and the risk of possible infections of pathogen to humans through the use of such collagen products containing materials derived from livestock including those derived from oxhide has been latently pointed out. Therefore, in view of safety and amount of sources, collagen derived from fishes has been suddenly receiving public attention as materials for cosmetics and for food and it is becoming important to use fish collagen having a low denaturing temperature as a starting material for collagen gel. However, although the risk involved in using collagen derived from fishes is low, due to its low denaturing temperature, its heat stability as a material is often insufficient. Therefore, for a starting material for a cell carrier and for a medical material, fish collagen is considered to be disadvantageous as compared with collagen derived from livestock.
The above-mentioned problems in conventional collagen materials such as insufficient stretching property and insufficient strength have been hindering a wide application of common livestock-derived collagen to a cell carrier or a medical material. In addition, there has been no satisfactory method for manufacturing medical materials where heat stability at least at 37° C. is required by using fish collagen.